Anti-fibrotic effects of Cuscuta chinensis with in vitro hepatic stellate cells and a thioacetamide-induced experimental rat model

Abstract Context:Cuscuta chinensis Lam. (Convolvulaceae) has been used as a traditional herbal remedy for treating liver and kidney disorders. Objective: Anti-fibrotic effects of C. chinensis extract (CCE) in cellular and experimental animal models were investigated. Materials and methods: HSC-T6 cell viability, cell cycle and apoptosis were analysed using MTT assay, flow cytometry and Annexin V-FITC/PI staining techniques. Thioacetamide (TAA)-induced fibrosis model was established using Sprague Dawley rats (n = 10). Control, TAA, CCE 10 (TAA with CCE 10 mg/kg), CCE 100 (TAA with CCE 100 mg/kg) and silymarin (TAA with silymarin 50 mg/kg). Fibrosis was induced by TAA (200 mg/kg, i.p.) twice per week for 13 weeks. CCE and silymarin were administered orally two times per week from the 7th to 13th week. Fibrotic related gene expression (α-SMA, Col1α1 and TGF-β1) was measured by RT-PCR. Serum biomarkers, glutathione (GSH) and hydroxyproline were estimated by spectrophotometer using commercial kits. Results: CCE (0.05 and 0.1 mg/mL) and silymarin (0.05 mg/mL) treatment significantly (p < 0.01 and p < 0.001) induced apoptosis (11.56%, 17.52% for CCE; 16.50% for silymarin, respectively) in activated HSC-T6 cells, compared with control group (7.26%). Further, rat primary HSCs showed changes in morphology with CCE 0.1 mg/mL treatment. In in vivo studies, CCE (10 and 100 mg/kg) treatment ameliorated the TAA-induced altered levels of serum biomarkers, fibrotic related gene expression, GSH, hydroxyproline significantly (p < 0.05–0.001) and rescued the histopathological changes. Conclusions: CCE can be developed as a potential agent in the treatment of hepatofibrosis.


Introduction
Cuscuta chinensis Lam. (Convolvulaceae), originally from China, has been used as an herbal medicine in several Asian countries for centuries. In Traditional Chinese Medicine (TCM), the semen of C. chinensis has been used as a tonic and aphrodisiac to improve sexual potency, prevent abortion and to enhance liver and kidney conditions (Donnapee et al. 2014). Pharmacologically, C. chinensis possess neuroprotective (Zhen et al. 2006), hepatoprotective, antioxidant (Yen et al. 2007), osteoblastogenic (Yang et al. 2009), genoprotective activities (Szeto et al. 2011) and improve renal function in experimental rats (Shin et al. 2011). Although C. chinensis showed a broad range of biological activities, there is no scientific evidence regarding the anti-fibrotic effects.
Hepatofibrosis results from chronic damage to the liver in conjunction with the accumulation of extracellular matrix (ECM) proteins, which is a characteristic of most types of chronic liver diseases (Friedman 2003). Hepatic fibrosis was historically thought to be a passive and irreversible process due to the collapse of the hepatic parenchyma and its substitution with a collagen-rich tissue (Schaffner and Klion 1968;Popper and Uenfriend 1970). Hepatic fibrosis is associated with activation of hepatic stellate cells (HSCs), the major source of the ECM proteins and is also caused by frequent hepatic injury with sustained inflammation in liver tissue and organ failure (Bruck et al. 2001;Henderson and Iredale 2007).
HSCs are considered as key participants in liver fibrosis development which is central process of fibrosis as the major source of fibrillary and non-fibrillar matrix protein (Iredale et al. 1998;Abramovitch et al. 2011). HSCs are usually quiescent cells, but in response to liver injury they undergo an activation process in which they become highly proliferative and synthesize a fibrotic matrix rich in type I collagen (Reeves and Friedman 2002). The phenotypic changes seen in activated HSCs often characterized as 'myofibroblastic activation' lead to excessive deposition of ECM and disrupt the normal architecture of the liver causing liver fibrosis, liver cirrhosis and liver cancer (Friedman 2003;Tsukada et al. 2006;Yoon et al. 2016). Therefore, it is important to induce the apoptosis of HSCs or prevent the secretion of the ECM by HSCs (Lee et al. 2014). Thus, in the present study, we investigated the antifibrotic effects of C. chinensis extracts (CCEs) in an in vitro system using HSC-T6 cells and an in vivo system using thioacetamide (TAA)-induced liver fibrosis rat model.

Plant material and extraction
C. chinensis, collected during May 2016, was purchased from Jecheon Chinese Medicinal Plant Co., Jecheon, South Korea and was authenticated by Prof. Jong-Bo Kim, a taxonomist at Konkuk University, South Korea, based on its microscopic and macroscopic characteristics. A voucher specimen (CC-KU2016) was kept in our department herbarium for future reference. For extraction, the dried semen of C. chinensis (300 g) was ground to a fine powder and extracted with 1 L ethanol (95%) using Soxhlet's extraction technique for three days at room temperature. The extract was then concentrated in a vacuum under reduced pressure and lyophilized. The final yield of the lyophilized CCE was 9.5% (w/w) and was stored at 4 C. The lyophilized powder of CCE was dissolved in 10% dimethyl sulphoxide (DMSO) and then filtered through a 0.22 lM syringe filter and stored as stock until use for each experiment. The final concentration of DMSO used for the study was not more than 0.1%.

Cell lines and culture
An immortalized rat's hepatic stellate cell lines (HSC-T6) were generously provided by Prof. Chang-Gue Son (Korean Hospital of Daejeon University, South Korea). HSC-T6 were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% FBS, 1% antibiotic-antimycotic in a humidified atmosphere of 5% CO 2 at 37 C. Chang liver cell line was purchased from ATCC (Manassas, VA). Chang liver cell line was used as a normal human cell line derived from normal liver tissue. The cells were cultured in DMEM (GIBCO, Carlsbad, CA) supplemented with 10% foetal bovine serum (FBS, GIBCO, Carlsbad, CA), 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA) in a humidified atmosphere of 5% CO 2 at 37 C. For activation, HSC-T6 cells were serum starved before treatment with CCE.

Primary HSCs isolation and culture
HSCs were isolated from 7-week-old male Sprague Dawley (SD) rats by in situ with pronase, collagenase, DNase perfusion and single-step Histogenz gradient as previously reported (Knook et al. 1982;Hendriks et al. 1985). Isolated HSCs were cultured in low glucose DMEM (GIBCO, Carlsbad, CA) containing 10% FBS (GIBCO, Carlsbad, CA) and 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA) on uncoated plastic maintained in a humidified atmosphere of 5% CO 2 at 37 C and these activated HSCs were used in the experiments. The growth medium was changed on a daily basis for seven days.

Cell cycle analysis
HSC-T6 cells (15 Â 10 5 cells/well) were cultivated in DMEM medium containing 10% FBS (GIBCO, Carlsbad, CA) and 1% antibiotic-antimycotic (GIBCO, Carlsbad, CA) maintained in a humidified atmosphere of 5% CO 2 at 37 C. Growth medium was changed on a daily basis for seven days. Sample materials were evaluated at CCE 0.05 and 0.1 mg/mL concentrations for 24 h at 37 C in an atmosphere of 5% CO 2 and 95% humidity. After 24 h, the cells were washed with PBS twice, suspended in 1 mL cold PI solution (50 lg/mL PI and 100 lg/mL RNase A). Then, the cells were incubated on ice for 30 min in the dark and then analysed with a flow cytometer.

Apoptosis analysis
Apoptosis was determined by Annexin V-FITC and PI (FICS Annexin V apoptosis Detection Kit I, BD Biosciences, Franklin Lakes, NJ). The processes of detection were carried out according to manufacturer's instruction. Data analysis was performed with CellQuest software (Beckton Dickinson, Franklin Lakes, NJ), which allowed assessing of specific population only. Individualization by gates was done according to size, granularity and fluorescent parameters. Both early apoptotic (Annexin V þ and PI À ) and late apoptotic (Annexin V þ and PI À ) cells were included in cell death determinations.

Animals and experiment design
Fifty specific-pathogen-free SD male rats (six-weeks old, 190-210 g) were purchased from a commercial animal breeder (Orient Bio, Seoul, Korea). Animals were housed in conventional cages under control conditions of temperature (23 ± 3 C), relative humidity (50 ± 20%) and 12 h light/dark cycle. After 1 week of acclimation, the rats were divided randomly into five groups of 10 animals each: Normal, TAA (TAA only), CCE 10 (TAA with CCE 10 mg/kg), CCE 100 (TAA with CCE 100 mg/kg) and positive control silymarin group (TAA with 50 mg/kg silymarin). Liver fibrosis was induced using a previously described procedure with slight modifications (Wallace et al. 2015). Briefly, TAA (200 mg/kg) was administered intraperitoneally (i.p.) twice a week for 13 weeks to four groups except normal group (injected normal saline, i.p.). CCE (10 or 100 mg/kg), silymarin (50 mg/kg) or distilled water was given by gastric gavage six times per week from the 7th week to the 13th week. Body weight was recorded once a week. After last CCE or silymarin administration, animals were fasted for 18 h, and then blood was collected from cardiac puncture under CO 2 anaesthesia. A portion of liver tissue stored at À80 C separately was used for hydroxyproline, GSH, protein expression determination. Liver tissue fixed in Bouin's solution was processed for histomorphological findings. A small portion of liver tissue fixed in RNAlater solution was stored at À80 C for gene expression studies. All animal experiments were approved by the Committee of Laboratory Animals according to the institutional guidelines of Konkuk University, South Korea (IACUC No. KU15017).

Serum biochemical analysis
Blood was collected through cardiac puncture under CO 2 anaesthesia on the final day of the experiments. Serum was separated using centrifugation (3000Âg, 15 min). Following the blood clotting, the serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) were determined using a GOT-GTP assay kit (Asan Pharmaceutical, Anseong-si, Korea).

Estimation of total GSH content
Total GSH was determined according to the method of Evans and Ellman (1959). Briefly, duplicate 50 lL aliquots of the samples (or GSH standard) were combined with 80 lL of a previously prepared DTNB. NADPH mixture (10 lL 4 mM DTMB and 70 lL 0.3 mM NADPH) is taken in a 96-well plate. Finally, 20 lL (0.06 U) of GSH reductase solution was added to each well and the absorbance was ensured at 405 nM after 5 min.

Determination of hydroxyproline in liver tissues
Hydroxyproline determination was performed using a light modification in the previous method (Takayama et al. 2003). Briefly, liver tissues (156 mg) stored at À70 C were homogenized in 1 mL of 6 N HCl and incubated overnight at 100 C. After passage of the acid hydrolysates through filter paper (Toyo Roshi Kaisha, Tokyo, Japan), 50 lL samples or hydroxyproline standards in 6 N HSL were air-dried. The dried samples were dissolved in methanol (50 lL), and then 1.2 mL of 50% isopropanol and 200 lL of chloramine-T solution were added to each followed by incubation at room temperature for 10 min. Ehrlich's solution (1.3 mL) was added, and the samples were incubated at 50 C for 90 min. The optical density of the reaction product was read at 558 nM using a spectrophotometer (Tecan, Morrisville, NC). A standard curve was constructed using serial dilutions of 0.1 mg/mL hydroxyproline solution.

Histopathology of liver tissue
Bouin's solution fixed liver tissues were embedded in paraffin and cut into 5 lM thick section for histomorphological examination. After drying, liver tissue section slides were stained with haematoxylin and eosin (H&E) and Masson's trichrome. For semiquantitative analysis of collagen expression, the blue-stained areas in Masson's trichrome stained sections were measured on an image analyser (ImageJ, NIH, Bethesda, MD).

Statistical analysis
The results are expressed as the mean ± standard error of the mean (S.E.M., n ¼ 10). Statistical analysis was carried out using Student's t-test using Graph Pad Prism software version 4.00 (Graph Pad Software Inc., San Diego, CA). Differences between groups were analysed by one-way analysis of variance (ANOVA). The values of p < 0.05 were considered as statistically significant.

Cell viability assay and primary HSC morphology
As shown in Figure 1(A), CCE was treated at various concentrations (0.01, 0.05, 0.1, 0.5, and 1.0 mg/mL) in Chang liver cell and HSC-T6 cells. CCE treated at indicated concentrations (0.05 and 0.1 mg/mL) did not exhibit any significant changes in the overall cell viability or produce toxicity in Chang liver cells and HSC-T6 cells. However, concentrations greater than 0.5 mg/mL showed significant effect on cell viability. Further the solvent, DMSO (0.1%) alone used for dissolving the CCE also did not show significant toxicity. Therefore, all in vitro experiments were performed with CCE 0.05 and/or 0.1 mg/mL as the concentrations were considered nontoxic and effective (Figure 1(A)).
As shown in Figure 1(B), untreated activated HSCs showed normal morphology (7th day). CCE (0.1 mg/mL) treatment for 24 h on eight day-cultured primary HSCs showed changes in cell morphology such as shrinking collagen fibre and cell degradation (Figure 1(C)). This indicated that CCE treatment influenced the morphology of the cultured activation HSCs by decreasing the number of viable HSCs and stretched fibres when compared with the non-treated activated HSCs after 24 h.

Cell cycle analysis
Flow cytometric analysis of CCE (0.05 and 0.1 mg/mL) treated HSC-T6 cells showed 1.74 and 2.62%, respectively, in the sub-G1 phase compared with non-treated cells showing a distribution of 1.06%. Silymarin was used as a positive control, and showed 1.68% of the cells in the sub-G1 phase. These results indicated that CCE treatment has mild effects including the induction of apoptosis in HSC-T6 cells (Figure 2).

Apoptosis analysis on HSC-T6 cells
As shown in Figure 3, HSC-T6 cells were treated with silymarin (0.05 mg/mL) and CCE (0.05 and 0.1 mg/mL) for 24 h.

Serum biochemical analysis
As shown in Figure 4(A,B), TAA-induced group significantly (p < 0.001) increased the AST and ALT serum levels. However, the levels of AST and ALT were significantly (p < 0.5$p < 0.001) decreased by CCE (10 and 100 mg/kg) treatment compared to TAA group. Especially, the AST value of CCE100 group was decreased about half compared to TAA group. Silymarin treatment also showed a positive trend with significant effect (p < 0.001).

Total glutathione contents in TAA-induced liver tissues
TAA treatment significantly (p < 0.001) reduced the total GSH contents in the liver tissues compared to normal group. CCE treated groups restored the TAA-induced decrease in total GSH significantly (p < 0.01). CCE 100 group showed superior effect in restoring the total GSH level compared with silymarin treated group (Figure 4(C)).

Determination of hydroxyproline in TAA-induced liver tissues
The hydroxyproline levels of TAA group significantly (p < 0.001) increased compared to control group in TAA-induced liver tissues. CCE treatment groups at both doses (10 and 100 mg/kg) Figure 1. Cell viability assay on Chang liver/HSC-T6 cells and morphological changes in primary HSCs on treatment with CCE. (A) Chang liver and HSC-T6 cells were incubated with CCE at indicated concentrations for 24 h and the cell viability was determined by MTT assay. (B) Primary HSCs were cultivated for 1 week and exposed to the CCE 0.1 mg/mL for 24 h (C). Pictures were taken after 24 h treatment with CCE. Magnification was 100Â. Arrows indicate HSCs. The data are expressed as means ± S.E.M. (n ¼ 10), using one-way analysis of variance (ANOVA) followed by Student's t-test. #p < 0.05, compared with control group. NS: not significant compared with control group.  attenuated this increase significantly (p < 0.05 and p < 0.01) compared with TAA group. CCE 100 group decreased hydroxyproline level same as to standard silymarin group (Figure 4(D)).

Histopathology of liver tissues
In the liver sections, control group showed normal morphology ( Figure 5(A)). TAA treatment showed abnormal liver pattern with formation of numerous nodules ( Figure 5(B)). However, CCE 100 and silymarin treatment markedly attenuated these changes ( Figure 5(C,D)). No obvious changes were observed in CCE 10 compared with TAA-induced group (Figure 5(E)). Masson's trichrome showed severe collagen accumulation (blue staining part) in the TAA group when compared with control group (Figure 6(A,B)), while the silymarin and CCE 100 group remarkably protected against collagen accumulation (Figure 6(C,D)). No remarkable changes were observed in CCE 10 group (Figure 6(E)). Fibrosis percentage area revealed significant damage in TAA treated group compared with control group (p < 0.001). However, CCE at both concentrations ameliorated these changes significantly (p < 0.05 for CCE 10 and p < 0.01 for CCE 100 group). Silymarin exhibited significant attenuating affect (p < 0.001) when compared with TAA treated group (Figure 6(F)).

Liver fibrosis related gene analysis in TAA-induced tissues
As shown in Figure 7, TAA treatment increased the gene expression of TGF-b, Col1a1, and a-SMA significantly (p < 0.001). However, CCE at both concentrations and silymarin decreased the gene expression of TGF-b, Col1a1 and a-SMA ( Figure  7(A-C)). Silymarin treated group exhibited superior effects when compared with CCE treated group in downregulating the TGF-b expression while showed similar effects when compared with CCE 100 group in inhibiting Col1a1, and a-SMA expression.

Discussion
Despite considerable medical advances, liver disorders, including hepatofibrosis, remain clinically elusive with no satisfactory treatment and cure. It was well documented TCMs are currently the world's most effective treatment for various liver disorders including cirrhosis, fibrosis and hepatitis (Dwivedi et al. 1991;Chen et al. 2010). In the present study, the Chinese traditional herb, C. chinensis was investigated for treating hepatofibrosis in cellular and experimental fibrotic rat model.
It is well known that HSCs activation and over expression is the key initial event in the pathogenesis of hepatofibrosis (Wang et al. 2000). Activated HSCs were also responsible for secreting collagen scar tissue, which can lead to liver cirrhosis (Morigi et al. 2004). When round-shape quiescent HSCs undergo Figure 4. Effect of CCE on AST/ALT levels, total glutathione (GSH) contents and hydroxyproline levels in TAA-induced liver fibrosis rats. (A) HSC-T6 cells were incubated with CCE and silymarin for 24 h. Levels of AST (A) and ALT (B) in serum were measured using spectrophotometry. Total GSH contents (C) and hydroxyproline levels (D) in liver tissues were measured using spectrophotometry. TAA (200 mg/kg): thioacetamide-induced liver fibrosis rats, silymarin (50 mg/kg): positive control rats, CCE 100: CCE 100 mg/kg treated rats, CCE 10: CCE 10 mg/kg treated rats. The data are expressed as means ± S.E.M. (n ¼ 10) using one-way analysis of variance (ANOVA) followed by Student's t-test. #p < 0.05 as compared with control group, Ã p < 0.05, ÃÃ p < 0.01, ÃÃÃ p < 0.001 as compared with TAA group.  (n ¼ 10) using one-way analysis of variance (ANOVA) followed by Student's t-test. #p < 0.05 as compared with control group, Ã p < 0.05, ÃÃ p < 0.01 and ÃÃÃ p < 0.001 as compared with TAA group. TAA: thioacetamide-induced liver fibrosis rats, silymarin: positive control rats, CCE 100: CCE 100 mg/kg treated rats and CCE 10: CCE 10 mg/kg treated rats. activation by liver damage, the production of ECM is increased, and their shape changes resembling myofibroblasts (Kisseleva and Brenner 2008). Further, activated HSCs are characterized by high density of collagen around scar cell and proliferation, contractility with increased ECM productions. Thus, morphological changes in the lipid droplets that decrease stretching fibres mean the inhibition of activated HSCs. Our data suggested that CCE treatment significantly inhibited the activation and altered the morphology of HSCs.
HSCs apoptosis plays a critical role in the spontaneous recovery from fibrosis (Elsharkawy et al. 2005;Henderson and Iredale 2007). Reduced formation of procollagen and increased ECM degradation via HSCs apoptosis may benefit in steady recovery from chronic liver fibrosis. In the present study, CCE induced apoptosis in HSC-T6 cells analysed by Annexin V and PI staining technique. Further, CCE (0.1 mg/mL) downregulated the gene expression of TGF-b, a-SMA and Col1a1, selective markers of HSCs activation in vitro.
TAA is a carcinogen which is known to produce marked hepatotoxicity in experimental animals (M€ uller et al. 1988;Wallace et al. 2015). One of the major changes in TAA-induced liver damage is the altered liver enzymes such as AST and ALT which are secreted into the blood. These are the most commonly used markers of hepatocyte injury (Johnston 1999). The levels of these markers can give a general indication of whether a disorder is acute or chronic and whether it is intra-or extra-hepatic liver damage (McClatchey 2002). In the present study, the TTAinduced increase in the levels of AST and ALT enzymes was significantly reduced by CCE treatment indicating that CCE might possess beneficial property for treating hepatic injury and fibrosis.
Further, TAA-induced liver damage also produces reactive oxygen species (ROS) leading to alteration of liver biological function marker, GSH which plays a major role as a reductant in oxidation-reduction processes (Carlberg and Mannervik 1985). Therefore, restoring GSH content might be helpful in TAAinduced oxidative liver damage. In the present study, the decreased levels of GSH in TAA-induced rats were restored with CCE treatment significantly (p < 0.01). Hydroxyproline is a posttranslational modification product of proline hydroxylation catalysed by an enzyme polyhydroxylase which is the main factor in collagen stabilization (Krane 2008;Palfi and Perczel 2008). Therefore, we determined hydroxyproline levels in liver tissues to confirm whether CCE would decrease hydroxyproline levels in TAA-induced liver tissues. TAA-induced significant increase in the hydroxyproline levels and CCE at both concentrations significantly ameliorated this increase in rat liver tissues.
TGF-b has multiple profibrogenic but also anti-inflammatory and immunosuppressive effects. The balance of these actions is required for maintaining tissue homeostasis and an aberrant expression of TGF-b is involved in a number of disease processes particularly in liver disorders (Gressner et al. 2002). TGF-b is produced by Kupffer cells and HSCs upregulate the transcription of the collagen genes (Col1a1 and Col1a2), which are observed in damaged liver and highly expressed in activated HSCs from cirrhotic liver (Jakowlew et al. 1991). Since the activated HSCs could promote the production of collagen, we also tested the effect of CCE on the expression of Col1a1 gene. An actin isoform, a-SMA is a specific marker for smooth muscle cell differentiation. Therefore, a-SMA expression has been used to identify activated HSCs that show a myofibroblastic phenotype (Carpino et al. 2005). In this report, we demonstrated that CCE significantly suppressed the TAA-induced increase in the expression of TGF-b, col1a1 and a-SMA in rat liver tissues. These results indicate that CCE exerts its antifibrotic action by inhibiting HSCs proliferation and activation.
Silymarin, the positive control used in this study, is well known for its beneficial role in liver disorders based on its antioxidant effects (Saller et al. 2001;Wu et al. 2009). In agreement, in the present study, silymarin protected TAA-induced hepatofibrosis both in vitro (0.5 mg/mL) and in vivo (50 mg/mL), however, the effects were inferior when compared with CCE at higher concentrations (0.1 mg/mL treated group in vitro and CCE 100 mg/ kg in vivo).
Earlier reports revealed that antioxidants, such as flavonoids and glycosides (Kawada et al. 1998), can effectively inhibit the proliferation of HSCs. The active constituents of C. chinensis include flavonoids, alkaloids, saccharides, lignan and resin glycosides (Yahara et al. 1994;Miyahara et al. 1996;Du et al. 1998;Ye et al. 2002). Some of the compounds isolated from C. chinensis have been suggested to be responsible for the various pharmacological activities including anti-oxidant activity (Chen et al. 2004) and anti-inflammatory activities (Liao et al. 2013). The compounds present in the CCE might act individually or synergistically in delivering such potent anti-hepatofibrotic effects. Therefore, isolation of single active constituents present in CCE exploring the detailed mechanism is quite necessary.

Conclusions
CCE showed hepatoprotective effects both in vitro and in vivo as evidenced by the increased apoptosis, inhibited ECM accumulation and decreased collagen in HSC-T6 cells. Further, CCE attenuated the TAA-induced changes in various parameters in in vivo liver fibrosis rat model. Our findings suggest that CCE may be developed as an effective therapeutic agent against various liver related disorders including liver fibrosis.